1. Field of the Invention
The present invention relates to a method for mining microbial genomes for genes that kill, or inhibit the growth of, various bacteria. The present invention also relates to genes and gene products that can be used as antibiotics or as tools for molecular biology.
2. Related Art
Microbes (bacteria, archaea, fungi and viruses) frequently produce and secrete compounds aimed at killing other microbes which help them in their continuous struggle for survival in their ecological niche. Such compounds can be small molecule antibiotics, such as the ones produced by various Streptomyces species [Watve, Arch Microbiol. 2001 November; 176(5):386-90], or proteinacious antibiotics, often known as bacteriocins [Riley & Wertz, Annu Rev Microbiol. 2002; 56: 117-37]. Microbes also produce non-secreted defense molecules that help them escape predation by viruses. For example, “abortive infection” genes are suicidal genes that are activated in some bacteria once they sensed they were infected by a virus. These genes lead to the death of the infected bacterium and, hence, to the survival of the surrounding bacterial community [Chopin, Curr Opin Microbiol. 2005 August; 8(4):473-9]. Viruses also frequently produce molecules that inhibit the growth of, or kill, microbial cells by various mechanisms such as degradation of RNA [Sanson, FEMS Microbiol Rev. 1995 August; 17(1-2):141-50], cell lysis [Schuch, Nature. 2002 Aug. 22; 418(6900):884-9] etc.
Proteins that target bacteria have a broad medical and biotechnological application spectrum. They can be used as direct antibiotics for human and veterinary medicine [Gillor 2005, Curr Pharm Des. 2005; 11(8):1067-75], as growth enhancers in livestock [Brashears, 2003. J. Food Prot. 66, 748-754], as food preservatives [Delves-Broughton, Antonie Van Leeuwenhoek. 1996 February; 69(2): 193-202], as genes engineered into probiotic bacteria [Gillor 2005, Curr Pharm Des. 2005; 11(8):1067-75], as killers of phytopathogenic bacteria for crop management [Penyalver 2000, Eur. J. Plant Pathol. 106, 801-810], etc.
One of the popular methods to study the function of a given gene is to clone it into a model bacterial species (with Escherichia coli (E. coli) being the most popularly used model) and to study the expressed product. However, gene products that are toxic to bacteria will usually be unclonable in E. coli due to their negative effect on the bacterial growth. As described below, the present invention provides a method for identifying regions from microbial genomes that are unclonable into E. coli, retrieve antimicrobial genes that reside in these regions, and demonstrate their toxicity to E. coli and other pathogenic microbes. The method relies on an improvement of the microbial genome sequencing process described in URL:<http://www.jgi.doe.gov/sequencing/strategy.html> and links therein.
One aspect of the present invention involves mapping of sequencing clones onto a “finished” (fully sequenced) microbial genomic sequence. Such mapping was noted to be beneficial for detection of toxic proteins also by Roberts (U.S. Patent Application Publication No. US 2006/0014179 A1), who searched for gaps in clone start sites on either sides of open reading frames, and inferred that such open reading frames can encode for toxic genes or endonucleases.